Supplementary Information

Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel

Ian Sofian Yunusa, Julian Wichmannb, Robin Wördenweberb, Kyle J. Lauersenb, Olaf Kruseb

and Patrik R. Jonesa*

aDepartment of Life Sciences, Imperial College London, SW7 2AZ London, UKbBielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec),

Universitätsstrasse 27, 33615, Bielefeld, Germany.

*Corresponding author: p.jones@imperial.ac.uk

IndexSupplementary Table 1. The inventory of materials ((A) plasmids, (B) linkers, (C) primers, (D) DNA parts) used to prepare DNA constructs for the creation of Synechocystis strains.

Supplementary Table 2. All Synechocystis sp. PCC 6803 strains used in the study.

Supplementary Table 3. All DNA constructs used for engineering Chlamydomonas (optimized transgenes, vector backbone, reporters, selection markers, NCBI accession numbers)

Supplementary Figure 1. Growth properties of engineered cyanobacteria strains. (A) The final OD730 and biomass accumulation of the engineered strains on day 10 when samples were extracted. (B) Representative growth profile for the first three strains shown in (A).

Supplementary Figure 2. Graphic illustration of all (key) Chlamydomonas DNA constructs used in the study

Supplementary Figure 3. The inactivation of aas (acyl-ACP synthase).

Supplementary Figure 4. GC-MS chromatogram for 7-heptadecene in native Chlamydomonas.

Supplementary Figure 5. Western blot with proteins extracted from engineered Chlamydomonas strains. (A) OleTJE strains, (B) CrFAP strains.

Supplementary Figure 6. Fluorescence microscopy with Chlamydomonas OleTJE and CrFAP strains.

Supplementary Figure 7. Polar and neutral lipid profiles in TesA-overexpressing Chlamydomonas strains.

Supplementary Figure 8. GC-MS chromatograms of suspected alkenes in 6803-FAP strains.

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Note that all of the supplementary information is presented in the order of mention in the main text.

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Supplementary Table 1. The inventory of materials ((A) plasmids, (B) linkers, (C) primers, (D) DNA parts) used to prepare DNA constructs for the creation of Synechocystis strains.

(A) Plasmid DNA used for transformation of cyanobacteriaAssembly Prefix

Linker Plasmid Suffix Linker

Plasmid generated Relevant Information

1

1MP pIY67 1S

pIY370

pIY370 is a plasmid storage containing rrnB1 T1 terminator and T7Te

terminator, gentamicin selection marker, and downstream region of

slr1311 site.

1P pIY127 2S2P pIY353 2MS

2MP pIY21 1MS

2

1MP pIY370 1S

pIY380

pIY380 is a plasmid used for chromosomal integration targeting the

slr1311 site with PpsbA2S as a promoter. This plasmid contains a GFP

dropout gene.

1P pIY23 2S2P pIY352 2MS

2MP pIY24 1MS

3LRBS1-5P pIY72 1S

pIY397 pIY397 is used to integrate ‘tesA in slr1311 site under PpsbA2S promoter.1P pIY380 LRBS1-5S

4

1MP pIY128 1S

pIY140

pIY140 is an RSF1010 overexpression plasmid with Pclac143 promoter and erythromycin as a selection marker. This plasmid contains a GFP dropout

gene.

1P pIY51 2S2P pIY102 2MS

2MP pIY24 1MS

5

1MP pIY128 1S

pIY417

pIY417 is an RSF1010 overexpression plasmid with Pcoa promoter and

erythromycin as a selecton marker. This plasmid contains a GFP dropout

gene.

1P pIY51 2S2P pPB345 2MS

2MP pIY24 1MS

6LRBS1-5P pIY72 1S

pIY397 pIY397 is used to integrate ‘TesA in slr1311 site under PpsbA2S promoter.1P pIY380 LRBS1-5S

7LRBS1-5P pIY72 1S

pIY196pIY196 is a ‘TesA overexpression

plasmid with Pclac143 promoter and erythromycin selection marker.1P pIY140 LRBS1-5S

8LRBS1-3P pIY395 1S

pIY402pIY402 is an UndB overexpression

plasmid with Pclac143 promoter and erythromycin selection marker.1P pIY140 LRBS1-3S

91P pIY67 2S

pIY608pIY608 is an empty RSF1010 plasmid

with Pclac143 promoter and erythromycin selection marker.2P pIY140 1S

101P pIY67 2S

pIY606pIY606 is an empty RSF1010 plasmid

with Pcoa promoter and erythromycin selection marker.2P pIY417 1S

11LRBS1-4P pIY619 LRBS2-4S

pIY625 pIY602 is used to overexpress ‘TesA and FAP under Pcoa promoter.LRBS2-4P pIY499 1S

1P pIY417 LRBS1-4S

12LRBS1-4P pIY619 LRBS2-4S

pIY630pIY630 is used to overexpress ‘TesA

and truncated FAP under Pcoa promoter.

LRBS2-4P pIY505 1S1P pIY417 LRBS1-4S

13LRBS1-5P pIY354 1S

pIY440pIY440 is used to overexpress ‘TesA,

Sfp, and CAR under Pclac143 promoter.1P pIY140 LRBS1-5S

14LRBS1-4P pIY619 1S

pIY679 pIY679 is used to overexpress ‘TesA under Pcoa promoter.1P pIY417 LRBS1-4S

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(B) DNA linkers used for preparation of constructs used to engineer cyanobacteria

Adapter Linker Mixed Prefix Linker (P linker)Name Sequence (5' to 3') Name Sequence (5' to 3')

1P-A TTTATTGAACTA 1P-L GGACTAGTTCAATAAATACCCTCTGACTGTCTCGGAG 1P2P-A TTCTTATTACCT 2P-L GGACAGGTAATAAGAACTACACGACTGGATACTGACT 2P

3P-A TGTTATTACAGA 3P-L GGACTCTGTAATAACAATACCGATAAAGCAACGAGTG 3P

5P-A TTGATTTATCCT 5P-L GGACAGGATAAATCAACTCGTAAGCAATACTGTCTGT 5P

LRBS1-5P-A ATTAGTGGAGGTTA LRBS1-5P-L GGACTAACCTCCACTAATTTACAACTGATACTTACCTGA LRBS1-5P

LRBS1-4P-A ATCACAAGGAGGTA LRBS1-4P-L GGACTACCTCCTTGTGATTTACAACTGATACTTACCTGA LRBS1-4P

LRBS1-3P-A AAAGAGGAGAAATA LRBS1-3P-L GGACTATTTCTCCTCTTTTTACAACTGATACTTACCTGA LRBS1-3P

LRBS2-4P-A ATCACAAGGAGGTA LRBS2-4P-L GGACTACCTCCTTGTGATTTTCTGCTACCCTTATCTCAG LRBS2-4P

1MP-A TCTGGTGGGT/iMe-dC/TCT 1MP-L GGACAGAGACCCACCAGATAATAGTGTTTCCACGAAGTG 1MP

2MP-A AACTTCGGAATC 2MP-L GGACGATTCCGAAGTTACACCAGATTGGACTGTTATTAC 2MP

Adapter Linker Mixed Suffix Linker (S linker)Name Sequence (5' to 3') Name Sequence (5' to 3')

1S-A TGTCGTAAGTAA 1S-L CTCGTTACTTACGACACTCCGAGACAGTCAGAGGGTA 1S2S-A TTTCACACCGAT 2S-L CTCGATCGGTGTGAAAAGTCAGTATCCAGTCGTGTAG 2S

3S-A TAGTGCCGTGAT 3S-L CTCGATCACGGCACTACACTCGTTGCTTTATCGGTAT 3S

5S-A GGCACTACTTCT 5S-L CTCGAGAAGTAGTGCCACAGACAGTATTGCTTACGAG 5S

LRBS1-5S-A GACGGTGTTCAA LRBS1-5S-L CTCGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAA LRBS1-5S

LRBS1-4S-A GACGGTGTTCAA LRBS1-4S-L CTCGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAA LRBS1-4S

LRBS1-3S-A GACGGTGTTCAA LRBS1-3S-L CTCGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAA LRBS1-3S

LRBS2-4S-A CCAATAGTAACA LRBS2-4S-L CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAA LRBS2-4S

1MS-A CGAGTTCTTACC 1MS-L CTCGGGTAAGAACTCGCACTTCGTGGAAACACTATTA 1MS

2MS-A CGATAGGT/iMe-dC/TCC 2MS-L nTATCGGTAATAACAGTCCAATCTGGTGT 2MS

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(C) DNA primers used for preparation of constructs used to engineer cyanobacteria

Primer Sequence (5’ to 3’)IY107 TCTGGTGGGTCTCTGTCCTTTACAGCTAGCTCAGTCCTAGIY108 CGATAGGTCTCCCGAGCCTGTGTGAAATTGTTATCCGCTCACIY113 GCACCAAGTACGGGAATTGGATTCGGTGIY114 CCGAGGTAATGGAGAGGACTAAGCACCAAGIY145 TCTGGTGGGTCTCTGTCCCCAGGCATCAAATAAAACGAAAGGCTCIY146 CGATAGGTCTCCCGAGCCCGGCCGGCAGGAGCAGAAGAGCATACIY155 CGATAGGTCTCCCGAGCCGTGGCAGCAGCCTAGGGAATTCTTACAGCIY211 TCTGGTGGGTCTCTGTCCGGCTTGATAGAACCTTACTTGGATGCCIY212 CGATAGGTCTCCCGAGCCAACTGACTAAACTTAGTCTAAAGGATTAATGAGIY213 TCTGGTGGGTCTCTGTCCTTCCTTGGTGTAATGCCAACTGAATAATCTGIY214 CGATAGGTCTCCCGAGCCGGCAATCGAGACAATTATTTTCCTCTAGACGIY238 TCTGGTGGGTCTCTGTCCATGGCGGACACGTTATTGATTCTGGIY258 TCTGGTGGGTCTCTGTCCATGGCGTCTGCTGTAGAAGATATTCGCAAAGIY259 CGATAGGTCTCCCGAGCCTTACGPB208 TCTGGTGGGTCTCTGTCCCTGCACTAAAGACAAGTGAGPB209 CGATAGGTCTCCCGAGCCGCTTTTTAACTTGGATTTTTACCPB78 TCTGGTGGGTCTCTGTCCAAGAGGAGAAAGGTACCATGGCGGACACGTTATTGATTCTGGPB79 CGATAGGTCTCCCGAGCCTTATGAGTCATGATTTACTAAAGGCTGCAACTGC

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(D) DNA parts used for preparation of constructs used to engineer cyanobacteria. All DNA parts were amplified from DNA templates using primers listed above (C) and stored in pJET1.2 blunt (Thermo Fischer).

Template Primer Plasmid Generated Plasmid Contained Relevant informationF R

pDF-laca IY145 IY146 pIY128 pJET-RSF1010 A plasmid storage containing a broad-host-range vector RSF1010 backbone

- - - pIY127 pJET-Gm A plasmid storage containing gentamicin cassette. A gift from Dr. Geoff Baldwin (Imperial College)

pPB223 IY107 IY108 pIY102 pJET-lacI-Pclac143 A plasmid storage containing a lac repressor and Pclac143 promoter

- - - pIY24 pJET-GFP A plasmid storage containing a superfold green fluorescence protein. GFP was ordered as a gBlock.

pET-TPC2b PB78 PB79 pIY72 pJET-’TesA A plasmid storage containing ‘TesA

pET-TPC2b PB78 IY155 pIY354 pJET-TPC2 A plasmid storage containing ‘TesA, Sfp, and CAR.

- - - pIY51 pJET-Ery A plasmid storage containing erythromycin cassette. Erythromycin cassette was ordered as a gBlock.

6803-WT genomic DNA IY211 IY212 pIY352 pJET-slr1311_up A plasmid storage containing the upstream region of slr1311.

6803-WT genomic DNA IY213 IY214 pIY353 pJET-slr1311_down A plasmid storage containing the downstream region of slr1311.

6803-WT genomic DNA PB208 PB209 pPB345 pJET-Pcoa A plasmid storage containing CoaR and Pcoa promoter.

- - - pIY395 pJET-UndB A plasmid storage containing UndB. UndB was ordered as a gBlock from IDT DNA.

- - - pIY499 pJET-FAP A plasmid storage containing FAP. FAP was ordered as a gBlock from IDT DNA.

pIY499 IY258 IY259 pIY505 pJET-‘FAP A plasmid storage containing truncated FAP.

- - - pIY21 pMB1-Kan A plasmid backbone harbouring ColE1 ori and a kanamycin selection marker.

- - - pIY23 pMB1-Amp A plasmid backbone harbouring ColE1 ori and a ampicillin selection marker.

- - - pIY67 pJET-termB15 A plasmid storage containing rrnB T1 terminator and T7Te terminator.

pIY72 IY238 PB79 pIY619 pJET-‘TesA A plasmid storage containing ‘TesA.aGuerrero et al., Plos ONE, 2012 bAkhtar et al., PNAS, 2013

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Supplementary Table 2. All Synechocystis sp. PCC 6803 strains used in the study.

Strain Relevant informationWT Synechocystis sp. PCC 6803 wild-type strain obtained from Prof. Klaas Hellingwerf (Univ of Amsterdam)aas Synechocystis sp. PCC 6803 acyl-ACP synthetase deletion strain.aas-‘TesA aas harbouring chromosomal integration of ‘tesA under PpsbA2S promoter. This strain was obtained by transforming plasmid pIY397.aas-‘TesA-1010-UndB aas-‘TesA carrying UndB overexpression RSF1010 plasmid pIY402.aas-1010-‘TesA aas carrying ‘TesA overexpression RSF1010 plasmid pIY196 using a Pclac143 promoteraas-1010-TPC2 aas carrying ‘TesA, Sfp, and CAR overexpression RSF1010 plasmid pIY440 using a Pclac143 promoteraas-1010-Pcoa-‘TesA aas carrying ‘TesA overexpression RSF1010 plasmid pIY679 using a Pcoa promoteraas-1010-Pcoa-‘TesA-FAP aas carrying ‘TesA and FAP overexpression RSF1010 plasmid pIY625 using a Pcoa promoteraas-1010-Pcoa-‘TesA-‘FAP aas carrying ‘TesA and truncated FAP overexpression RSF1010 plasmid pIY630 using a Pcoa promoter

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Supplementary Table 3. All constructs used for engineering Chlamydomonas.

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Supplementary Figure 1. Growth properties of engineered cyanobacteria strains. (A) The final OD730 and biomass accumulation of the engineered strains on day 10 when samples were extracted. (B) Representative growth profile for the first three strains shown in (A).

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Supplementary Figure 2. Graphic illustration of all (key) Chlamydomonas DNA constructs used in the study. Vector constructs for nuclear transgene expression from C. reinhardtii. The base pOpt2_YFP_Paro vector (Wichmann et al 2018) was used to construct chloroplast targeted protein products using the photosystem I reaction center subunit II (PsaD) chloroplast targeting peptide (CTP). All other constructs were fused to create protein products with C-terminal mVenus (YFP) or mRuby2 (RFP) reporters. Nuclear expression constructs all contain the rbcs2 i1 intron in copies spread throughout their sequence (blue boxes). The fluorescent reporters contain rbcs2 i2 as previously described (Lauersen et al 2015). E. coli thioesterase A’ (TesA), Jeotgalicoccus sp. terminal olefin-forming fatty acid decarboxylase (OleTJE) and its fusion to Rhodococcus sp. P450 reductase RhFRED. C. reinhardtii native fatty acid photodecarboxylase (CrFAP) was synthetized with its own native CTP (*), but was also cloned with the PsaD CTP instead and expressed as YFP or RFP fusions, as well as in fusion with the TesA construct mediated by the modular nature of the pOpt2 vectors. All vectors use the common HSP70-RBCS2-i1 fusion promoter and corresponding 3’ UTR and either paromomycin (Paro) or zeocin/bleomycin (Ble) resistance cassettes from the pOpt2 vectors (Wichmann et al. 2018). * indicates site of additional glycine to add BamHI site and allow cloning removal of the native chloroplast targeting peptide and replacement with the PsaD CTP. Paro – all vectors confer paromomycin resistance except the single zeocin (Ble) resistance vector. The vector numbers are shown to the right.

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Supplementary Figure 3. Inactivation of aas. (A) Schematic diagram of acyl-ACP synthetase (encoded by slr1609) knockout. (B) Colony PCR analysis of Δaas and the wild-type strain with positive and negative controls using primers IY113 and IY114. (C) Image of the wild-type (left) and Δaas (right) strains after 10 days of cultivation. Circle highlight visible precipitates forming in the Δaas culture. Representative chromatograms from extracted (D) Δaas and (E) wild-type cultures. C15:0 pentadecanoic acid was used as internal standard.

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Supplementary Figure 4. Polar and neutral lipid profiles in the wild-type control (UVM4) and TesA-overexpressing Chlamydomonas strains under nitrogen replete (+N) and deficient (-N) conditions. Extraction of total lipids, separation into neutral and polar lipids, transesterification and GC-MS analysis were performed to determine relative lipid profiles for each strain under each condition. The left section of the graph displays quantities for C8-C12 FAMEs only, using a different axis.

(A) Polar lipds.

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(B) Neutral lipds.

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Supplementary Figure 5. Western blot to determine full-length fusion protein expression from the nuclear genome of C. reinhardtii.

(A) OleTJE strains. Proteins were extracted from 2 day old cultures in logarithmic phase. An anti-GFP HRP linked polyclonal antibody (Thermo Fisher Scientific) was used for detection of YFP-fused proteins on nitrocellulose membranes. Ponceau S staining of the membrane is shown as a protein loading control. M – PageRuler Prestained protein ladder (Thermo). P – UVM4 parental strain.

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(B) CrFAP and CrFAP-TesA fusion expressing strains. Same methods as above. * indicates native CrFAP chloroplast targeting peptide construct (vector Cr7).

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Supplementary Figure 6. Fluorescence microscopy with Chlamydomonas strains expressing various OleT and CrFAP constructs. Wide-field fluorescence microscopy was used to confirm chloroplast localization of YFP-linked constructs as previously described (Lauersen et al 2016). The over-expressed proteins, from vectors listed in Supp. Table 3 (above), in each strain is listed on the right, and the vector numbers are shown on the left.

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Supplementary Figure 7. GC-MS chromatogram for 7-heptadecene in wild-type Chlamydomonas. Identification of 7-heptadecene (7-h) and product partitioning between cells, culture medium (supernatant), and a dodecane overlay. A Chlamydomonas reinhardtii strain UVM4 was cultivated in TAP medium for 5 d in constant light with and without an overlay of 5% dodecane. (A) Only traces of the product were detectible in the overlay, and the major fraction was found in the cell pellet (middle panel and C). α-humulene served as internal standard (IS). 7-heptadecene was detected at earlier retention times than the commercial 1-heptadecene (1-h) standard (lower panel), however, it showed (B) similar mass fractionation pattern. (C) Presence (+d) or absence (-d) of the dodecane overlay did not affect total product yields. Error bars represent 95% confidence intervals of strains cultivated in biological triplicate.

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Supplementary Figure 8. Unidentified alkenes accumulating in the 6803-aas-'TesA-FAP strain. Two unique (relative to wild-type) products that accumulated in the 6803-aas-'TesA-'FAP strain are most likely also alkenes based on NIST mass spectrometry (2.2) analysis of individual peaks eluting at 6.86 ((A) 6,9-heptadecadiene) and 6.88 minutes ((B) 8-heptadecene), respectively (indicated by the arrow).

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